RNA was isolated from cells and tissues using the mRNeasy Kit (Qiagen) or Trifast (PeqLab) and reverse-transcribed into cDNA using High Capacity cDNA Reverse Transcription Kit (Fermentas) according to manufacturer's protocols. Expression of mRNA was measured using FastSybrGreen Mix (Applied Biosystems) in a StepOne RT-PCR 7,500 system (Applied Biosystems). Ct-values of target genes were normalized to mHPRT and expressed as fold mean WT control using efficiencies calculated by LinReqPCR16 (link) for each amplification. The following primers were used:HPRT: for: CGCAGTCCCAGCGTCGTG; rev: CCATCTCCTTCATGACATCTCGAG; PTEN: for: ACACCGCCAAATTTAACTGC; rev: TACACCAGTCCGTCCCTTTC; IL-10: for: AGCTGAAGACCCTCAGGATG; rev: TGGCCTTGTAGACACCTTGG; Arg1: for: GGAAAGCCAATGAAGAGCTG; rev: GCTTCCAACTGCCAGACTGT; Fizz: for: CTGGATTGGCAAGAAGTTCC; rev: CCCTTCTCATCTGCATCTCC; Stab1: for: CCCTCCTTCTGCTCTGTGTC; rev: CAAACTTGGTGTGGATGTCG; IL-23a: for: ATGCTGGATTGCAGAGCAGT; rev: ACGGGGCACATTATTTTTAG; Ym1: for: TTTCTCCAGTGTAGCCATCCTT; rev: TCTGGGTACAAGATCCCTGAA.
Stepone rt pcr 7 500 system
Manufactured by Thermo Fisher Scientific
The StepOne RT-PCR 7,500 system is a real-time PCR instrument designed for gene expression analysis and quantification. It provides accurate and reliable data for a wide range of applications, including gene expression profiling, genotyping, and pathogen detection.
Automatically generated - may contain errors
2 protocols using stepone rt pcr 7 500 system
1
Real-Time qPCR Expression Analysis
2
Quantitative RT-PCR Analysis of Colonic Immune Genes
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RNA was isolated from colon lamina propria single cell suspension using Trifast (PeqLab) and reverse-transcribed into cDNA using High Capacity cDNA Reverse Transcription Kit (Fermentas) according to manufacturer’s protocols. Expression of mRNA was measured using FastSybrGreen Mix (Applied Biosystems) in a StepOne RT-PCR 7500 system (Applied Biosystems). Ct-values of target genes were normalized to mHPRT and expressed as fold mean WT control using efficiencies calculated using LinReqPCR (15 (link)) for each amplification. The following primers were used:
hprt: for: CGCAGTCCCAGCGTCGTG; rev: CCATCTCCTTCATGACATCTCGAG;
il10: for: AGCTGAAGACCCTCAGGATG; rev: TGGCCTTGTAGACACCTTGG;
ifng: for: TGAGCTCATTGAATGCTTGG; rev: ACAGCAAGGCGAAAAAGGAT,
il17a; for: TGAGCTTCCCAGATCACAGA; rev: TCCAGAAGGCCCTCAGACTA,
foxp3: for: GCGAAAGTGGCAGAGAGGTA; rev: TCCAAGTCTCGTCTGAAGGC,
il6: for: CAAGTCGGAGGCTTAATTACACATG; rev: ATTGCCATTGCACAACTCTTTTCT.
hprt: for: CGCAGTCCCAGCGTCGTG; rev: CCATCTCCTTCATGACATCTCGAG;
il10: for: AGCTGAAGACCCTCAGGATG; rev: TGGCCTTGTAGACACCTTGG;
ifng: for: TGAGCTCATTGAATGCTTGG; rev: ACAGCAAGGCGAAAAAGGAT,
il17a; for: TGAGCTTCCCAGATCACAGA; rev: TCCAGAAGGCCCTCAGACTA,
foxp3: for: GCGAAAGTGGCAGAGAGGTA; rev: TCCAAGTCTCGTCTGAAGGC,
il6: for: CAAGTCGGAGGCTTAATTACACATG; rev: ATTGCCATTGCACAACTCTTTTCT.
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